Cultured cells catabolize certain nutrients including carbon sources, amino acids, and vitamins. The cells undergo cell division and produce useful cellular products such as monoclonal antibodies, as well as waste products such as ammonia and lactic acid. However, the accumulation of waste products and depletion of nutrients limits the functional life-span of a cell culture. In batch culture, cell death occurs after minimal levels of nutrients have been exhausted and/or intolerable levels of waste products have accumulated. Various solutions to the build-up of waste products and depletion of nutrients have been attempted.
Continuous growth of cultures may be achieved by removing a portion of the culture and replacing it with fresh medium at regular intervals. This serves to remove waste products and replenish needed nutrients. In this method, fluid containing cells and media is continuously withdrawn from the cell culture container and replaced with fresh medium. Alternatively, in a prefusion system cells may be confined to the cell culture vessel such that only medium is withdrawn while fresh medium is added.
However, the components of a cell culture system are not depleted uniformly. Thus, although the perfused media may be depleted of three or four nutrients, it may contain adequate amounts of other components. The removal of these useful components by perfusion is uneconomical.
In these various systems, nutrient and waste levels are maintained by withdrawing "spent" medium and replacing with fresh medium. However, especially in large scale/high density cultures, perfusion of nutrients requires the costly transfer of large volumes of media from the manufacturer to the culture facility, into the bioreactor, and finally to the product isolation facility. One way to minimize the cost and inconvenience of shipping large volumes of media is by the use of concentrates of tissue culture media. However, concentrates need to be reconstituted with water prior to addition to the cultures and large volumes of spent media are generated.
Perfusion is associated with other disadvantages. For example, perfusion systems are susceptible to plugging by cells which must be separated from spent culture media. This is a significant concern for high density cultures since prodigious amount of fluids must be exchanged, sometimes greater than one culture volume per day. In addition, cell products must be isolated from the large volumes of spent culture media generated, and after products are isolated, large volumes of spent culture media must be disposed of.
Absorption technology has been advocated for removal of the waste product ammonia (Gordon, A., Greenbaum, M., Marantz, L., MacArthur, M. and Maxwell, M., "A Sorbent Based Low Volume Recirculating Dialysate System", Trans. Amer. Soc. Artif. Int. Organs 15:347-352, 1969)). Although this system eliminates ammonia, nutrient levels are not affected. In addition, in-place regeneration of the adsorptive agent, zirconium phosphate, is difficult after the agent is saturated with ammonia. Reverse osmosis systems again offer only toxin reduction capabilities with no means of increasing nutrient levels.
Thus, it would be an obvious benefit to the practice of cell culture if nutrients and waste products could be maintained within desired levels without the need for perfusion of large volumes of media.